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Supplementary Figure 2. Percent radioiodine uptake by region, all patients.

Supplementary Table 1. Summary of Patient Study Data.

Supplementary Figure 1. Patients with new iodine uptake after four weeks of dabrafenib.

Supplementary Table 2. Spot Urine Iodide Measurements.

supplemental figure legend

Supplementary Table 3. Serum TSH values.

Purpose: To determine whether the selective BRAF inhibitor, dabrafenib, can stimulate radioiodine uptake in BRAF V600E-mutated unresectable or metastatic iodine-refractory papillary thyroid cancer (PTC).Experimental Design: Ten patients with BRAF V600E-mutant iodine-refractory PTC were enrolled. Absence of radioiodine uptake on iodine-131 whole body scan obtained within 14 months of study entry was required. Each patient received dabrafenib (150 mg twice daily) for 25 days before thyrotropin α-stimulated iodine-131 whole body scan (4 mCi/148 MBq). Patients whose scan showed new sites of radioiodine uptake remained on dabrafenib for 17 more days, and then were treated with 150 mCi (5.5 GBq) iodine-131. The primary endpoint of the study was the percentage of patients with new radioiodine uptake after treatment with dabrafenib.Results: Six of 10 patients (60%) demonstrated new radioiodine uptake on whole body scan after treatment with dabrafenib. All 6 were treated with 5.5 GBq iodine-131. Two patients had partial responses and 4 patients had stable disease on standard radiographic restaging at 3 months. Thyroglobulin decreased in 4 of 6 treated patients. One patient developed squamous cell carcinoma of the skin. There were no other significant adverse events attributed to dabrafenib.Conclusions: Dabrafenib can stimulate radioiodine uptake in patients with metastatic BRAF V600E-mutant iodine-refractory PTC, representing a potential new therapeutic approach for these patients. Clin Cancer Res; 21(5); 1028–35. ©2014 AACR.

Mitochondrial electron transport chain (ETC) dysfunction due to mutations in the nuclear or mitochondrial genome is a common cause of metabolic disease in humans and displays striking tissue specificity depending on the affected gene. The mechanisms underlying tissue-specific phenotypes are not understood. Complex I (cI) is classically considered the entry point for electrons into the ETC, and in vitro experiments indicate that cI is required for basal respiration and maintenance of the NADsup+/sup/NADH ratio, an indicator of cellular redox status. This finding has largely not been tested in vivo. Here, we report that mitochondrial complex I is dispensable for homeostasis of the adult mouse liver; animals with hepatocyte-specific loss of cI function display no overt phenotypes or signs of liver damage, and maintain liver function, redox and oxygen status. Further analysis of cI-deficient livers did not reveal significant proteomic or metabolic changes, indicating little to no compensation is required in the setting of complex I loss. In contrast, complex IV (cIV) dysfunction in adult hepatocytes results in decreased liver function, impaired oxygen handling, steatosis, and liver damage, accompanied by significant metabolomic and proteomic perturbations. Our results support a model whereby complex I loss is tolerated in the mouse liver because hepatocytes use alternative electron donors to fuel the mitochondrial ETC.Mitochondria are specialised structures inside cells that help to convert nutrients into energy. They take electrons from nutrients and use them to power biochemical reactions that supply chemical fuel. Previous studies of cells grown in the laboratory have found that electrons enter this process via a large assembly of proteins in mitochondria called complex I. Understanding the mechanism of energy production is important, as issues with mitochondria can lead to a variety of metabolic diseases. However, it is still unclear how complex I acts in living animals. Lesner et al. addressed this knowledge gap by genetically removing a key protein from complex I in the liver of mice. Surprisingly, the animals did not develop any detectable symptoms and maintained healthy liver function. Mice did not seem to compensate by making energy in a different way, suggesting that complex I is not normally used by the mouse liver for this process. This research suggests that biologists should reconsider the mechanism that mitochondria use to power cells in animals. While the role of Complex I in electron transfer is well established in laboratory-grown cells and some organs, like the brain, it cannot be assumed this applies to the whole body. Understanding energy production in specific organs could help researchers to develop nutrient-based therapies for metabolic diseases.

SUMMARYA metabolic hallmark of cancer identified by Warburg is the increased consumption of glucose and secretion of lactate, even in the presence of oxygen. Although many tumors exhibit increased glycolytic activity, most forms of cancer rely on mitochondrial respiration for tumor growth. We report here that Hürthle cell carcinoma of the thyroid (HTC) models harboring mitochondrial DNA-encoded defects in complex I of the mitochondrial electron transport chain exhibit impaired respiration and alterations in glucose metabolism. CRISPR/Cas9 pooled screening identified glycolytic enzymes as selectively essential in complex I-mutant HTC cells. We demonstrate in cultured cells and a PDX model that small molecule inhibitors of lactate dehydrogenase selectively induce an ATP crisis and cell death in HTC. This work demonstrates that complex I loss exposes fermentation as a therapeutic target in HTC and has implications for other tumors bearing mutations that irreversibly damage mitochondrial respiration.

A phenotypic high-throughput screen identified a benzamide small molecule with activity against small cell lung cancer cells. A "clickable" benzamide probe was designed that irreversibly bound a single 50 kDa cellular protein, identified by mass spectrometry as β-tubulin. Moreover, the anti-cancer potency of a series of benzamide analogs strongly correlated with probe competition, indicating that β-tubulin was the functional target. Additional evidence suggested that benzamides covalently modified Cys239 within the colchicine binding site. Consistent with this mechanism, benzamides impaired growth of microtubules formed with β-tubulin harboring Cys239, but not β3 tubulin encoding Ser239. We therefore designed an aldehyde-containing analog capable of trapping Ser239 in β3 tubulin, presumably as a hemiacetal. Using a forward genetics strategy, we identified benzamide-resistant cell lines harboring a Thr238Ala mutation in β-tubulin sufficient to induce compound resistance. The disclosed chemical probes are useful to identify other colchicine site binders, a frequent target of structurally diverse small molecules.

The outcomes of patients with SCLC have not yet been substantially impacted by the revolution in precision oncology, primarily owing to a paucity of genetic alterations in actionable driver oncogenes. Nevertheless, systemic therapies that include immunotherapy are beginning to show promise in the clinic. Although, these results are encouraging, many patients do not respond to, or rapidly recur after, current regimens, necessitating alternative or complementary therapeutic strategies. In this review, we discuss ongoing investigations into the pathobiology of this recalcitrant cancer and the therapeutic vulnerabilities that are exposed by the disease state. Included within this discussion, is a snapshot of the current biomarker and clinical trial landscapes for SCLC. Finally, we identify key knowledge gaps that should be addressed to advance the field in pursuit of reduced SCLC mortality. This review largely summarizes work presented at the Third Biennial International Association for the Study of Lung Cancer SCLC Meeting.

SummaryMitochondrial electron transport chain (ETC) dysfunction due to mutations in the nuclear or mitochondrial genome is a common cause of metabolic disease in humans, and displays striking tissue specificity depending on the affected gene. The mechanisms underlying tissue specific phenotypes are not understood. Complex I (cI) is classically considered the entry point for electrons into the ETC, and in vitro experiments indicate that cI is required for basal respiration and maintenance of the NAD+/NADH ratio, an indicator of cellular redox status. This finding has largely not been tested in vivo. Here, we report that mitochondrial complex I (cI) is dispensable for homeostasis of the adult mouse liver; animals with hepatocyte-specific loss of cI function display no overt phenotypes or signs of liver damage, and maintain liver function, redox and oxygen status. Further analysis of cI-deficient livers did not reveal significant proteomic or metabolic changes, indicating little to no compensation is required in the setting of complex I loss. In contrast, complex IV (cIV) dysfunction in adult hepatocytes results in decreased liver function, impaired oxygen handling, steatosis, and liver damage, accompanied by significant metabolomic and proteomic perturbations. Metabolomic analysis suggests that the electron transfer flavoprotein complex constitutes a major route for electron entry into the hepatic ETC. Our results support a model whereby complex I loss is tolerated in the mouse liver because hepatocytes use alternative electron donors to fuel the mitochondrial ETC.

Ewing sarcoma (EWS) is a pediatric malignancy driven by the EWSR1-FLI1 fusion protein formed by the chromosomal translocation t(11; 22). The small molecule TK216 was developed as a first-in-class direct EWSR1-FLI1 inhibitor and is in phase II clinical trials in combination with vincristine for patients with EWS. However, TK216 exhibits anti-cancer activity against cancer cell lines and xenografts that do not express EWSR1-FLI1, and the mechanism underlying cytotoxicity remains unresolved. We apply a forward-genetics screening platform utilizing engineered hypermutation in EWS cell lines and identify recurrent mutations in TUBA1B, encoding ⍺-tubulin, that prove sufficient to drive resistance to TK216. Using reconstituted microtubule (MT) polymerization in vitro and cell-based chemical probe competition assays, we demonstrate that TK216 acts as an MT destabilizing agent. This work defines the mechanism of cytotoxicity of TK216, explains the synergy observed with vincristine, and calls for a reexamination of ongoing clinical trials with TK216.

Mitochondria play an essential role in cellular metabolism, generation of reactive oxygen species (ROS), and the initiation of apoptosis. These properties enable mitochondria to be crucial integrators in the pathways of tumorigenesis. An open question is to what extent variation in the mitochondrial genome (mtDNA) contributes to the biological heterogeneity observed in human tumors. In this review, we summarize our current understanding of the role of mtDNA genetics in relation to human cancers.

Dabrafenib, a BRAF selective tyrosine kinase inhibitor (TKI) has been associated with pancreatitis alone, and in combination with trametinib (a MEK pathway inhibitor). We report a case of dabrafenib induced acute pancreatitis in a patient undergoing treatment for metastatic papillary thyroid cancer (PTC). This adverse event has not been previously reported in thyroid cancer patients using dabrafenib. Case: A 61-year-old female with papillary thyroid cancer status post total thyroidectomy with pathology revealing right 7.6cm PTC with extrathyroidal extension and 13/72 metastatic lymph nodes. She received 100mCi of radioactive iodine (RAI). Ten months post-surgery, she underwent revision neck dissection with 3/36 nodes positive for metastatic disease and neck muscle invasion by PTC. Molecular testing demonstrated AKT1 and BRAFV600E mutations. Due to progressive neck and mediastinal tumors, recommendation was to proceed with systemic therapy. Initially started dabrafenib monotherapy; after 3 doses patient developed burning epigastric pain and fever. Examination revealed abdominal tenderness, elevated lipase at 2266 U/L (8-76U/L), mild hyperbilirubinemia and leukocytosis. CT abdomen/pelvis revealed peripancreatic fat stranding consistent with focal acute pancreatitis. No history of alcohol use. Dabrafenib was assessed to be culprit of pancreatitis; with immediate discontinuation of TKI and supportive management patient improved. At present patient is tolerating Lenvatinib therapy. BRAFV600E mutation occurs in 29-83% of thyroid cancers affecting growth, proliferation, survival, migration, and loss of tissue-specific differentiation via the intracellular MEK/ERK pathway1. RAI offers an important treatment modality for thyroid cancer patients, however patients with BRAFV600E are often poor responders or refractory to RAI1. Dabrafenib, a specific BRAFV600E tyrosine kinase inhibitor in combination with trametinib (a MEK inhibitor) is FDA approved for treatment BRAFV600E mutated anaplastic thyroid cancer; furthermore there is evidence on PTC responses including redifferentiation 2. Pancreatitis has been reported in

Hurthle cell tumors (HCT), including Hurthle cell adenomas (HCA) and Hurthle cell carcinomas (HCCs), arise in the thyroid gland and are defined in part by an accumulation of mitochondria. These neoplasms were long considered a subtype of follicular neoplasm, although HCT is now generally considered a distinct entity. HCTs exhibit overlapping but distinct clinical features compared to follicular tumors, and several studies have demonstrated that HCTs harbor distinct genomic alterations compared to other forms of thyroid cancer. Two studies recently reported the most complete characterization of the HCC genome to date. These studies assessed complementary cohorts of HCC specimens. The study by Ganly et al. consisted of a large panel of primary HCCs, including 32 widely invasive and 24 minimally invasive primary tumors. Exome and RNA sequencing of material isolated from fresh-frozen tumor specimens was performed. The study by Gopal et al. utilized exome and targeted sequencing to characterize the nuclear and mitochondrial genomes of 32 primary tumors and 38 resected regional and distant metastases using DNA isolated from formalin-fixed paraffin-embedded tissues. Here, HCC is briefly reviewed in the context of these studies.

Despite the paucity of therapeutic advances in SCLC, considerable progress in understanding the biology, molecular biology, model systems and potential therapeutic targets has been made. Studies of early lung and neuroendrocrine cell development models have provided insights into the cell of origin for SCLC. New GEMMs have illustrated the universal importance of TP53 and RB1 gene mutations in the pathogenesis and the potential role of additional genetic changes as well as changes in transcription factor expression. PDXs and CDXs provide new means for preclinical testing of new therapies. Molecular studies have identified the high mutation burden found in SCLC and have identified differences between SCLC, carcinoids and large cell neuroendocrine tumors. Potential therapeutic targets including EZH2, PARP, CDK1, MCL1, BCL2, BIM, SHH (Sonic Hedgehog), WNT, NOTCH1, Aurora Kinase, FGFR, PIK3CA, RET, THZ1, JAK-STAT, FAK, CXCR4, PD-L1, Fuc-GM1, CD56 and CD47. Ongoing and future clinical trials have to show which of these candidates can be translated into an effective targeted therapy. Thus, the future of improving outcomes for SCLC patients appears promising but there are still a number of unanswered questions which need to be addressed in the future and these are outlined below. Small Cell Lung Cancer Major Questions of Translational Relevance What are the mechanisms underlying the universal development of chemoresistance? SCLC is in nearly every case very sensitive to platinum-etoposide chemotherapy with dramatic clinically beneficial responses. However, in nearly every case the tumors become resistant to this chemotherapy. What is the mechanism of this resistance, are there ways to avoid it, and what are additional therapies that could kill such resistant tumor cells? What are the mechanisms of highly metastatic behavior in SCLC? SCLC is in nearly every case highly metastatic from the time of clinical diagnosis. What are the most important mechanisms responsible for the metastatic behavior and can these be therapeutically targeted? A subset of this question, is that SCLC appear to be much enriched in cancer stem cells (“tumor initiating cells”, TICs) and does targeting stem cell signaling pathways provide effective therapy for primary and metastatic sites of SCLC? Are there therapeutic targets of “acquired vulnerability” associated with RB1 or TP53 mutations in SCLC? Tumor suppressor genes RB1 and TP53 abnormalities are essentially universal in SCLCs. Are there acquired vulnerabilities associated with changes in these two genes that can be therapeutically targeted? Do the many other mutations occurring in human SCLC (but not in GEMMs) provide therapeutic targets – “acquired vulnerabilities for human SCLC?” Current evidence indicates that human SCLCs have many other genetic (mutations) and epigenetic changes besides those involving the key oncogenic drivers (such as TP53, RB1, LMYC, NFIB). By contrast, mouse GEMM SCLC have very few additional changes. Presumable the differences in mutation rates result from exposure to cigarette carcinogens in humans but not in mice. Do the other mutations in human SCLC provide acquired vulnerabilities of therapeutic vulnerabilities, do any these represent “synthetic lethalities” with the main driver oncogene changes, and are there several vulnerabilities common across multiple SCLCs or are these vulnerabilities “private” and found in only individual tumors? Can we develop therapeutic strategies targeting important SCLC driver transcription factors? Several transcription factors appear to be very important in the growth and survival of SCLC including ASCL1, NeuroD1, myc family members, and SOX2). Are there therapeutic strategies that can be directed against these key transcription factors and do they provide quantitatively multiple logs of tumor cell kill to allow development of curative strategies? What are the important differences in human preclinical models of SCLC that influence discovery and validation of new therapies? Currently there are several types of human preclinical models of SCLC including SCLC lines, SCLC cell line xenografts (CDXs), patient derived SCLC xenografts (PDXs), and circulating SCLC tumor cells isolated from peripheral blood of SCLC patients. While these all share common genetic abnormalities found in SCLC (such as TP53, RB1, myc family members), we need to know what are the molecular differences between these different models and SCLC tumor samples and whether preclinical therapeutic responses are similar or different between these models. Can we continue to use all of the models for development of new therapies or do we need to only use one type of preclinical model? Are the differences between human SCLC and GEMM of SCLC that influence the discovery and validation of new therapies? Genetically engineered mouse models of lung cancer (GEMMs) appear to be very similar to human SCLC. However, it appears that mouse GEMM SCLC do not appear to be sensitive to platin-etoposide chemotherapy. What are the differences between human and the GEMM models of SCLC that explain this discrepancy? Are there important differences between human and GEMM preclinical models that could influence the discovery and validation of new SCLC therapies? What are the biologic reasons for SCLCs expressing ASCL1 vs NeuroD1 as a lineage oncogene? The majority of human SCLCs have ASCL1 as the major neuroendocrine lineage driver gene. However, a subset of human SCLCs express high levels of NeuroD1 and not ASCL1. Currently, there is no evidence that mouse lung neuroendocrine cells can express Neuro D1. This raises the question of whether small cell cancers predominantly expressing NeuroD1 arise in the human lung or in some other primary site. Do genetic abnormalities in histologically normal epithelium of SCLC patients provide diagnostic and chemopreventative targets? There appear to be a much greater frequency of genetic abnormalities in the histologically normal epithelium of patients with SCLC compared to those with NSCLC. Does this information provide ways for early diagnosis or implementation of chemoprevention strategies for SCLC using these genetic alterations as molecular biomarkers? Most recently the US National Cancer Institute released a Request for Application (RFA) (PAR-16-049, PAR-16-050, PAR-16-051) for grants specifically focusing on SCLC, and hopefully through these grants many of the above questions will be addressed.

Summary Target identification for biologically active small molecules remains a major barrier for drug discovery. Cancer cells exhibiting defective DNA mismatch repair (dMMR) have been used as a forward genetics system to uncover compound targets. However, this approach has been limited by the dearth of cancer cell lines that harbor naturally arising dMMR. Here, we establish a platform for forward genetic screening using CRISPR/Cas9 to engineer dMMR into mammalian cells. We demonstrate the utility of this approach to identify mechanisms of drug action in mouse and human cancer cell lines using in vitro selections against three cellular toxins. In each screen, compound-resistant alleles emerged in drug-resistant clones, supporting the notion that engineered dMMR enables forward genetic screening in mammalian cells.

Purpose: To determine whether the selective BRAF inhibitor, dabrafenib, can stimulate radioiodine uptake in BRAF V600E-mutated unresectable or metastatic iodine-refractory papillary thyroid cancer (PTC). Experimental Design: Ten patients with BRAF V600E-mutant iodine-refractory PTC were enrolled. Absence of radioiodine uptake on iodine-131 whole body scan obtained within 14 months of study entry was required. Each patient received dabrafenib (150 mg twice daily) for 25 days before thyrotropin α-stimulated iodine-131 whole body scan (4 mCi/148 MBq). Patients whose scan showed new sites of radioiodine uptake remained on dabrafenib for 17 more days, and then were treated with 150 mCi (5.5 GBq) iodine-131. The primary endpoint of the study was the percentage of patients with new radioiodine uptake after treatment with dabrafenib. Results: Six of 10 patients (60%) demonstrated new radioiodine uptake on whole body scan after treatment with dabrafenib. All 6 were treated with 5.5 GBq iodine-131. Two patients had partial responses and 4 patients had stable disease on standard radiographic restaging at 3 months. Thyroglobulin decreased in 4 of 6 treated patients. One patient developed squamous cell carcinoma of the skin. There were no other significant adverse events attributed to dabrafenib. Conclusions: Dabrafenib can stimulate radioiodine uptake in patients with metastatic BRAF V600E-mutant iodine-refractory PTC, representing a potential new therapeutic approach for these patients. Clin Cancer Res; 21(5); 1028–35. ©2014 AACR.

Summary Hurthle cell carcinoma of the thyroid (HCC) is a form of thyroid cancer recalcitrant to radioiodine therapy that exhibits an accumulation of mitochondria. We performed whole-exome sequencing on a cohort of primary, recurrent, and metastatic tumors, and identified recurrent mutations in DAXX, TP53, NRAS, NF1, CDKN1A, ARHGAP35, and the TERT promoter. Parallel analysis of mtDNA revealed recurrent homoplasmic mutations in subunits of complex I of the electron transport chain. Analysis of DNA copy-number alterations uncovered widespread loss of chromosomes culminating in near-haploid chromosomal content in a large fraction of HCC, which was maintained during metastatic spread. This work uncovers a distinct molecular origin of HCC compared with other thyroid malignancies.

The diagnosis of the follicular variant of papillary thyroid carcinoma (FVPTC) is increasingly common. Recent studies have suggested that FVPTC is heterogeneous and comprises multiple tumor types with distinct biological behaviors and underlying genetics.The purpose of this work was to identify the prevalence of mutations and gene fusions in known oncogenes in a panel representative of the common spectrum of FVPTC diagnosed at an academic medical center and correlate the clinical and pathological features obtained at the initial diagnosis with the tumor genotype.We performed SNaPshot genotyping on a panel of 129 FVPTCs of ≥1 cm for 90 point mutations or small deletions in known oncogenes and tumor suppressors and identified gene fusions using an anchored multiplex PCR assay targeting a panel of rearranged oncogenes.We identified a mutation or gene fusion in 70% (89 of 127) of cases. Mutations targeting the RAS family of oncogenes were the most frequently observed class of alterations, present in 36% (46 of 127) of cases, followed by BRAF mutation, present in 30% (38 of 127). We also detected oncogenic rearrangements not previously associated with FVPTC, including TFG-ALK and CREB3L2-PPARγ. BRAF mutation was significantly associated with unencapsulated tumor status.These data support the hypothesis that FVPTC is composed of distinct biological entities, with one class being identified by BRAF mutation and support the use of clinical genotyping assays that detect a diverse array of rearrangements involving ALK and PPARγ. Additional studies are necessary to identify genetic drivers in the 30% of FVPTCs with no known oncogenic alteration and to better predict behavior in tumors with known genotypes.

Small cell lung carcinoma (SCLC) is a highly lethal, smoking-associated cancer with few known targetable genetic alterations. Using genome sequencing, we characterized the somatic evolution of a genetically engineered mouse model (GEMM) of SCLC initiated by loss of Trp53 and Rb1. We identified alterations in DNA copy number and complex genomic rearrangements and demonstrated a low somatic point mutation frequency in the absence of tobacco mutagens. Alterations targeting the tumor suppressor Pten occurred in the majority of murine SCLC studied, and engineered Pten deletion accelerated murine SCLC and abrogated loss of Chr19 in Trp53; Rb1; Pten compound mutant tumors. Finally, we found evidence for polyclonal and sequential metastatic spread of murine SCLC by comparative sequencing of families of related primary tumors and metastases. We propose a temporal model of SCLC tumorigenesis with implications for human SCLC therapeutics and the nature of cancer-genome evolution in GEMMs, National Cancer Institute (U.S.) (K08CA160658)

Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity.

Thyroid cancer is the fifth most common cancer in women in the USA, and an estimated over 62 000 new cases occurred in men and women in 2015. The incidence continues to rise worldwide. Differentiated thyroid cancer is the most frequent subtype of thyroid cancer and in most patients the standard treatment (surgery followed by either radioactive iodine or observation) is effective. Patients with other, more rare subtypes of thyroid cancer-medullary and anaplastic-are ideally treated by physicians with experience managing these malignancies. Targeted treatments that are approved for differentiated and medullary thyroid cancers have prolonged progression-free survival, but these drugs are not curative and therefore are reserved for patients with progressive or symptomatic disease.

Genetically-engineered mouse models (GEMMs) of cancer are increasingly being utilized to assess putative driver mutations identified by large scale sequencing of human cancer genomes. In order to accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant EGFR, mutant Kras or by overexpression of MYC. Tumors from EGFR- and Kras- driven models exhibited respectively 0.02 and 0.07 non-synonymous mutations/megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras- driven models both exhibited recurrent whole chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared to human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity.

Introduction: Because small-cell lung carcinomas (SCLC) are seldom resected, human materials for study are limited. Thus, genetically engineered mouse models (GEMMs) for SCLC and other high-grade lung neuroendocrine (NE) carcinomas are crucial for translational research. Methods: The pathologies of five GEMMs were studied in detail and consensus diagnoses reached by four lung cancer pathology experts. Hematoxylin and Eosin and immunostained slides of over 100 mice were obtained from the originating and other laboratories and digitalized. The GEMMs included the original Rb/p53 double knockout (Berns Laboratory) and triple knockouts from the Sage, MacPherson, and Jacks laboratories (double knockout model plus loss of p130 [Sage laboratory] or loss of Pten [MacPherson and Jacks laboratories]). In addition, a GEMM with constitutive co-expression of SV40 large T antigen and Ascl1 under the Scgb1a1 promoter from the Linnoila laboratory were included. Results: The lung tumors in all of the models had common as well as distinct pathological features. All three conditional knockout models resulted in multiple pulmonary tumors arising mainly from the central compartment (large bronchi) with foci of in situ carcinoma and NE cell hyperplasia. They consisted of inter- and intra-tumor mixtures of SCLC and large-cell NE cell carcinoma in varying proportions. Occasional adeno- or large-cell carcinomas were also seen. Extensive vascular and lymphatic invasion and metastases to the mediastinum and liver were noted, mainly of SCLC histology. In the Rb/p53/Pten triple knockout model from the MacPherson and Jacks laboratories and in the constitutive SV40/T antigen model many peripherally arising non-small-cell lung carcinoma tumors having varying degrees of NE marker expression were present (non-small-cell lung carcinoma-NE tumors). The resultant histological phenotypes were influenced by the introduction of specific genetic alterations, by inactivation of one or both alleles of specific genes, by time from Cre activation and by targeting of lung cells or NE cell subpopulations. Conclusion: The five GEMM models studied are representative for the entire spectrum of human high-grade NE carcinomas and are also useful for the study of multistage pathogenesis and the metastatic properties of these tumors. They represent one of the most advanced forms of currently available GEMM models for the study of human cancer. Key Words: Neuroendocrine carcinomas; Small-cell lung carcinoma; Lung carcinoma; Non–small-cell lung cancer; Genetically engineered mouse models; Pathology

The basic helix-loop-helix transcription factors Hand1 and Hand2 display dynamic and spatially restricted expression patterns in the developing heart. Mice that lack Hand2 die at embryonic day 10.5 from right ventricular hypoplasia and vascular defects, whereas mice that lack Hand1 die at embryonic day 8.5 from placental and extra-embryonic abnormalities that preclude analysis of its potential role in later stages of heart development. To determine the cardiac functions of Hand1, we generated mice harboring a conditional Hand1-null allele and excised the gene by cardiac-specific expression of Cre recombinase. Embryos homozygous for the cardiac Hand1 gene deletion displayed defects in the left ventricle and endocardial cushions, and exhibited dysregulated ventricular gene expression. However, these embryos survived until the perinatal period when they died from a spectrum of cardiac abnormalities. Creation of Hand1/2 double mutant mice revealed gene dose-sensitive functions of Hand transcription factors in the control of cardiac morphogenesis and ventricular gene expression. These findings demonstrate that Hand factors play pivotal and partially redundant roles in cardiac morphogenesis, cardiomyocyte differentiation and cardiac-specific transcription.

Endothelin-A (ET(A)) is a G-protein-coupled receptor expressed in the neural crest-derived mesenchyme of the pharyngeal arches during craniofacial development. Targeted deletion of the ET(A) receptor or its ligand endothelin-1 (ET-1) causes cleft palate and hypoplasia of the mandible, otic cup, and tympanic ring. Previously we showed that Galpha(q)/Galpha(11)-null mice die around E11.0, whereas Galpha(q)((-/-))Galpha(11)((+/-)) mice survive to birth with hypomorphic phenotypes similar to, but less severe than, ET(A) or ET-1-null mice. To determine whether ET-1 signaling is transduced by Galpha(q)/Galpha(11) proteins, we examined the expression patterns of several ET-1 dependent and independent transcription factors in Galpha(q)/Galpha(11)-deficient embryos. Expression of genes encoding the ET-1-dependent transcription factors Dlx3, Dlx6, dHAND, and eHAND was specifically downregulated in the pharyngeal arches of Galpha(q)/Galpha(11)-deficient mice. In contrast, pharyngeal arch expression of the homeobox gene Msx1, which is not regulated by ET-1 signaling, was maintained in these embryos. We conclude that the Galpha(q) and Galpha(11) proteins serve as the intracellular mediators of ET-1 signaling in the pharyngeal arch mesenchyme.

Congenital heart disease in humans results from abnormal morphogenesis of the embryonic cardiovascular system. The characterization of mutations affecting cardiovascular development in animal models ranging from flies to mice has identified many of the key signaling molecules and transcriptional regulators of heart formation. Many of these molecules are also mutated in familial forms of human congenital heart disease. Through the use of animal models combined with analysis of human pedigrees, a molecular framework that controls formation of the vertebrate heart is beginning to emerge.

// Pierre Vanden Borre 1 , Viswanath Gunda 1 , David G. McFadden 2 , Peter M. Sadow 1 , Shohreh Varmeh 1 , Maria Bernasconi 1 and Sareh Parangi 1 1 Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 2 Thyroid Unit, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts Correspondence: Sareh Parangi, email: // Keywords : Thyroid, BRAF, SRC Received : April 3, 2014 Accepted : June 24, 2014 Published : June 26, 2014 Abstract Anaplastic (ATC) and refractory papillary thyroid cancer (PTC) lack effective treatments. Inhibition of either oncogenic BRAF or SRC has marked anti-tumor effects in mouse models of thyroid cancer, however, neither drug induces notable apoptosis. Here we report that the SRC-inhibitor dasatinib further sensitizes BRAF V600E -positive thyroid cancer cells to the BRAF V600E -inhibitor PLX4720. Combined treatment with PLX4720 and dasatinib synergistically inhibited proliferation and reduced migration in PTC and ATC cells. Whereas PLX4720 did not induce robust apoptosis in thyroid cancer cells, combined treatment with dasatinib induced apoptosis in 4 of 6 lines. In an immunocompetent orthotopic mouse model of ATC, combined PLX4720 and dasatinib treatment significantly reduced tumor volume relative to PLX4720 treatment alone. Immune cell infiltration was increased by PLX4720 treatment and this effect was maintained in mice treated with both PLX4720 and dasatinib. Further, combined treatment significantly increased caspase 3 cleavage in vivo relative to control or either treatment alone. In conclusion, combined PLX4720 and dasatinib treatment induces apoptosis, increases immune cell infiltration and reduces tumor volume in a preclinical model of ATC, suggesting that the combination of these FDA-approved drugs may have potential for the treatment of patients with ATC or refractory PTC.

Anaplastic thyroid carcinoma (ATC) has among the worst prognoses of any solid malignancy. The low incidence of the disease has in part precluded systematic clinical trials and tissue collection, and there has been little progress in developing effective therapies. v-raf murine sarcoma viral oncogene homolog B (BRAF) and tumor protein p53 (TP53) mutations cooccur in a high proportion of ATCs, particularly those associated with a precursor papillary thyroid carcinoma (PTC). To develop an adult-onset model of BRAF-mutant ATC, we generated a thyroid-specific CreER transgenic mouse. We used a Cre-regulated Braf(V600E) mouse and a conditional Trp53 allelic series to demonstrate that p53 constrains progression from PTC to ATC. Gene expression and immunohistochemical analyses of murine tumors identified the cardinal features of human ATC including loss of differentiation, local invasion, distant metastasis, and rapid lethality. We used small-animal ultrasound imaging to monitor autochthonous tumors and showed that treatment with the selective BRAF inhibitor PLX4720 improved survival but did not lead to tumor regression or suppress signaling through the MAPK pathway. The combination of PLX4720 and the mapk/Erk kinase (MEK) inhibitor PD0325901 more completely suppressed MAPK pathway activation in mouse and human ATC cell lines and improved the structural response and survival of ATC-bearing animals. This model expands the limited repertoire of autochthonous models of clinically aggressive thyroid cancer, and these data suggest that small-molecule MAPK pathway inhibitors hold clinical promise in the treatment of advanced thyroid carcinoma.

Neural crest cells play a key role in craniofacial development. The endothelin family of secreted polypeptides regulates development of several neural crest sublineages, including the branchial arch neural crest. The basic helix–loop–helix transcription factor dHAND is also required for craniofacial development, and in endothelin-1(ET-1) mutant embryos, dHAND expression in the branchial arches is down-regulated, implicating it as a transcriptional effector of ET-1 action. To determine the mechanism that links ET-1 signaling to dHAND transcription, we analyzed the dHANDgene for cis-regulatory elements that control transcription in the branchial arches. We describe an evolutionarily conserveddHAND enhancer that requires ET-1 signaling for activity. This enhancer contains four homeodomain binding sites that are required for branchial arch expression. By comparing protein binding to these sites in branchial arch extracts from endothelin receptor A(EdnrA) mutant and wild-type mouse embryos, we identified Dlx6, a member of the Distal-less family of homeodomain proteins, as an ET-1-dependent binding factor. Consistent with this conclusion,Dlx6 was down-regulated in branchial arches from EdnrAmutant mice. These results suggest that Dlx6 acts as an intermediary between ET-1 signaling and dHAND transcription during craniofacial morphogenesis.

Limb outgrowth and patterning of skeletal elements are dependent on complex tissue interactions involving the zone of polarizing activity (ZPA) in the posterior region of the limb bud and the apical ectodermal ridge. The peptide morphogen Sonic hedgehog (SHH) is expressed specifically in the ZPA and, when expressed ectopically, is sufficient to mimic its functions, inducing tissue growth and formation of posterior skeletal elements. We show that the basic helix-loop-helix transcription factor dHAND is expressed posteriorly in the developing limb prior to Shh and subsequently occupies a broad domain that encompasses the Shh expression domain. In mouse embryos homozygous for a dHAND null allele, limb buds are severely underdeveloped and Shh is not expressed. Conversely, misexpression of dHAND in the anterior region of the limb bud of transgenic mice results in formation of an additional ZPA, revealed by ectopic expression of Shh and its target genes, and resulting limb abnormalities that include preaxial polydactyly with duplication of posterior skeletal elements. Analysis of mouse mutants in which Hedgehog expression is altered also revealed a feedback mechanism in which Hedgehog signaling is required to maintain the full dHAND expression domain in the developing limb. Together, these findings identify dHAND as an upstream activator of Shh expression and important transcriptional regulator of limb development.

The basic helix-loop-helix (bHLH) transcription factors, Hand1 and Hand2 (refs 1,2), also called eHand/Hxt/Thing1 and dHand/Hed/Thing2 (refs 3,4), respectively, are expressed in the heart and certain neural-crest derivatives during embryogenesis. In addition, Hand1 is expressed in extraembryonic membranes, whereas Hand2 is expressed in the deciduum. Previous studies have demonstrated that Hand2 is required for formation of the right ventricle of the heart and the aortic arch arteries. We have generated a germline mutation in the mouse Hand1 gene by replacing the first coding exon with a beta-galactosidase reporter gene. Embryos homozygous for the Hand1 null allele died between embryonic days 8.5 and 9.5 and exhibited yolk sac abnormalities due to a deficiency in extraembryonic mesoderm. Heart development was also perturbed and did not progress beyond the cardiac-looping stage. Our results demonstrate important roles for Hand1 in extraembryonic mesodermal and heart development.

6014Background: ATC is the most aggressive type of thyroid cancer with a median survival of 5 months from diagnosis. ATC patients with genomic alterations (GA) in TSC2, ALK, BRAF show occasional, l...

The concept of prosthesis-directed implant-supported restoration is well accepted. The implementation of this principle for patients requesting full fixed implant-supported maxillary prosthetics has not been thoroughly described. We present a technique for the evaluation and preprosthetic surgical management of patients who are edentulous in the maxilla and wish to have fixed implant-supported crown and bridge prosthetics.

Purpose: To determine whether the selective BRAF inhibitor dabrafenib can stimulate radioactive iodine uptake in BRAF V600E mutated advanced papillary thyroid cancer (PTC) that is unable take up iodine. Patients and Methods: Ten patients with unresectable or metastatic PTC were enrolled. All had tumors with a BRAF V600E mutation and complete absence of radioiodine uptake on iodine-131 whole body scan obtained within 14 months of study entry. Each patient received dabrafenib (150 mg twice daily) for 3 weeks followed by thyrotropin alfa-stimulated iodine-131 whole body scan. If new iodine uptake occurred, dabrafenib was continued for an additional two weeks, followed by the administration of a therapeutic dose of iodine-131. Results: All ten patients completed the study. Dabrafenib resulted in new iodine-131 uptake in six of the ten patients. All six were treated with radioioactive iodine, leading to a complete response in one patient, a partial response in a second patient and stable disease in three patients. Four of six treated patients had decreases in serum thyroglobulin. No toxic effects of grade 2 or higher were attributable to dabrafenib. One patient developed a new squamous cell carcinoma of the skin which was successfully excised with clear margins. Conclusion: Dabrafenib produces increases in iodine uptake in patients with iodine-refractory advanced BRAF V600E mutant PTC and is well tolerated. Additional studies to determine efficacy are warranted. Citation Format: Stephen M. Rothenberg, David G. McFadden, Edwin L. Palmer, Gilbert H. Daniels, Lori J. Wirth. Dabrafenib stimulates radioiodine uptake in BRAF V600E mutant advanced papillary thyroid cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT234. doi:10.1158/1538-7445.AM2014-CT234

Despite that fact that 90% of cancer deaths are due to metastasis, relatively little is known about how metastases evolve from primary cancers. We subjected 101 trios consisting of primary tumor, brain metastasis, and normal reference tissue to whole exome sequencing (WES). To analyze the data, we developed novel computational tools to derive high quality allelic copy-number profiles directly from the WES data. These were used to perform an integrative analysis of somatic copy-number alterations (SCNAs) and somatic single nucleotide variants (SSNVs). This analysis allowed us to estimate the clonal architecture of the primary and metastatic samples from each patient, and to reconstruct a single phylogenetic tree relating all of the subclones detected in both samples. We determined that all of the matched metastases analyzed likely formed from a single clone. In all cases, we observed a sibling relationship between the metastasis and primary tumor samples, indicating that they share a common ancestor, but ongoing evolution in the primary tumor resulted in fully clonal mutations in the primary biopsy that were not present in the metastasis sample. An important corollary of this is that in no case was a metastasis-founding subclone detected in a primary tumor sample. Using mutational spectra associated with tobacco exposure in lung cancers, we determined that most of the evolution leading up to the brain metastasis like occurred within the lung. In contrast with these findings, analysis of related primary tumor and metastasis families in a mouse model of small cell lung cancer using identical methodology revealed evidence of more variable patterns of metastatic spread. Analysis of draining lymph nodes revealed that multiple subclones of the primary contributed to these metastases. In addition, we uncovered evidence for the sequential spread of metastases, consistent with multiregion sequencing of primary and metastatic human pancreatic cancer. The extension of this finding to a mouse cancer model suggests this may be a more frequent feature of cancer metastasis, and raises the possibility of genetically dissecting these processes in this experimental system. Citation Format: Scott L. Carter, Priscilla K. Brastianos, David G. McFadden, Thales Papagiannakopoulos, Amaro Taylor-Weiner, Kristian Cibulskis, Tyler Jacks, William Hahn, Gad Getz. Modes of metastasis evolution in human and murine cancer revealed by whole exome sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3992. doi:10.1158/1538-7445.AM2014-3992

The Hairy-related transcription-factor ( HRT ) genes encode three related basic helix–loop–helix transcription factors that show sequence similarity to the Hairy and Enhancer of split family of transcriptional repressors. HRT proteins are expressed in specific regions of the developing heart, vasculature, pharyngeal arches and somites, and the periodicity of their expression in somitic precursors mirrors that of Notch signaling-related molecules. In the present study, we show that the intracellular domain of the Notch1 receptor (Notch1 IC), which is constitutively active, up-regulates HRT2 expression in 10T½ fibroblasts. Luciferase reporter assays using the regulatory regions of the mouse HRT genes revealed that transcription of all three genes is stimulated by Notch1 IC. The promoters of the HRT genes share homology in a binding site for Suppressor of Hairless [Su(H)], a transcriptional mediator of Notch signaling. A dominant-negative Su(H) mutant abolished Notch-activated HRT2 expression, and mutation of the conserved Su(H) consensus site in the HRT2 promoter attenuated transcriptional activation by Notch. Ectopic expression of HRT proteins also blocked activation of HRT2 expression by Notch1 IC through a mechanism requiring the basic region, but not the conserved carboxyl-terminal YQPW-TEVGAF motif of HRT2. These findings identify HRT genes as downstream targets for Notch signaling and reveal a negative autoregulatory loop whereby HRT proteins repress their own expression through interference with Notch signaling.

Heart formation in vertebrates is believed to occur in a segmental fashion, with discreet populations of cardiac progenitors giving rise to different chambers of the heart. However, the mechanisms involved in specification of different chamber lineages are unclear. The basic helix-loop-helix transcription factor dHAND is expressed in cardiac precursors throughout the cardiac crescent and the linear heart tube, before becoming restricted to the right ventricular chamber at the onset of looping morphogenesis. dHAND is also expressed in the branchial arch neural crest, which contributes to craniofacial structures and the aortic arch arteries. Using a series of dHAND-lacZ reporter genes in transgenic mice, we show that cardiac and neural crest expression of dHAND are controlled by separate upstream enhancers and we describe a composite cardiac-specific enhancer that directs lacZ expression in a pattern that mimics that of the endogenous dHAND gene throughout heart development. Deletion analysis reduced this enhancer to a 1.5 kb region and identified subregions responsible for expression in the right ventricle and cardiac outflow tract. Comparison of mouse regulatory elements required for right ventricular expression to the human dHAND upstream sequence revealed two conserved consensus sites for binding of GATA transcription factors. Mutation of these sites abolished transgene expression in the right ventricle, identifying dHAND as a direct transcriptional target of GATA factors during right ventricle development. Since GATA factors are not chamber-restricted, these findings suggest the existence of positive and/or negative coregulators that cooperate with GATA factors to control right ventricular-specific gene expression in the developing heart.

Purpose: This study assessed the surgical complications after open treatment for fractures of the mandibular condylar process. Patients and Methods: A total of 178 patients with unilateral fractures of the mandibular condylar process, 85 treated closed and 93 treated open, were included in this study. A tabulation of surgical findings and intraoperative and postoperative complications was prospectively performed. Standardized animating facial photographs were obtained at several postsurgical intervals and were examined and scored by a prosthodontist and an orthodontist for signs of facial nerve palsy and the quality of the surgical scar. Standard statistical methods were used to assess differences between open and closed treatment groups. Results: There were very few intraoperative or postoperative complications. At the 6-week point, 17.2% of patients treated open had some weakness of their facial nerve. This had resolved by 6 months. The scars were judged either wide or hypertrophic in 7.5% of cases. Conclusions: Based on this study, surgical complications of open treatment of condylar process fractures that lead to permanent dysfunction or deformity are uncommon. © 2000 American Association of Oral and Maxillofacial Surgeons

Background: The MAPK and Src signaling pathways have emerged as critical drivers and mediators of papillary and anaplastic (PTC and ATC) thyroid cancer progression and the individual inhibition of either pathway has marked anti-tumor effects in mouse models. Unfortunately, neither drug induces apoptosis and drug resistance may develop when either is used. We postulated that the inhibition of the Src signaling pathway would further sensitize BRAFV600E-positive thyroid cancer cells to PLX4720 treatment. Methods: Proliferation and apoptosis were analyzed in four novel murine PTC and ATC cell lines derived from genetically engineered mice. Cells were treated with the BRAFV600E-inhibitor, PLX4720 (10μM), or the Src kinase inhibitor, dasatinib (50 nM), or a combination of the two drugs. Single and combinatorial dose response was determined and analyzed using Chou-Talalay. To determine the effect of combinatorial treatment in vivo, 105 murine ATC cells (TBP-3743 - BRAFV600E/WT; p53−/−) were orthotopically implanted in syngeneic immunocompetent mice. Mice were randomized into four groups and placed on treatment regimens 3 days following implantation, consisting of either control chow, PLX4720 chow (ab libitium), dasatinib gavage (50mg/kg) or a combination of PLX4720 chow and dasatinib gavage. Results: Basal pERK, pSrc(Y416) and pFAK(Y861) indicated active MAPK and Src signaling pathways in the thyroid cancer cell lines. PLX4720 treatment reduced pERK in the ATC lines, while dasatinib alone reduced both pSrc(Y416) and pFAK(Y861). The combination index (CI) of PLX4720 and dasatinib was calculated to be Conclusion: Combinatorial treatment of PTC and ATC cells with PLX4720 and dasatinib synergistically reduces proliferation and induces apoptosis in vitro and results in apoptosis and reduced tumor size relative to PLX4720 treatment alone in a pre-clinical mouse model of ATC. Together, these findings suggest that Src-inhibition enhances the anti-tumor effect of BRAFV600E-inhibition and the combination of these two FDA-approved drugs may have potential as a treatment paradigm for patients with ATC or refractory PTC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A141. Citation Format: Pierre Vanden Borre, Viswanath Gunda, David McFadden, Peter Sadow, Sareh Parangi. Dasatinib augments the antitumor effect of PLX4720 in a novel immunocompetent mouse model of BRAFV600E-positive anaplastic thyroid cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A141.

6025 Background: Resistance to radioactive iodine is a leading cause of mortality in differentiated thyroid carcinoma. The MAPK pathway is a major determinant of iodine uptake into thyroid carcinoma cells. Mutations in BRAF activate this pathway, resulting in resistance to radioactive iodine. A pilot study using the MEK1/2 inhibitor, selumetinib, (Ho, ASCO 2012) increased radioiodine uptake in a subset of thyroid cancers. Methods: This is a single institution, single arm pilot study investigating the potential for the BRAF inhibitor dabrafenib to induce radioiodine uptake in metastatic, BRAF-mutant, radioiodine-refractory papillary thyroid carcinoma (PTC). The primary endpoint is increased radioiodine uptake demonstrated on a 4mCi 131-I whole body scan. Patients with increased uptake receive 14 additional days of dabrafenib followed by treatment with 150mCi 131-I. Secondary endpoints include safety and tolerability and clinical benefit as measured by decreases in serum thyroglobulin and objective response rate per modified RESIST 1.1. Results: To date, 7 patients have been enrolled. All had negative 131-I scans within 14 months of enrollment. No dose adjustments for toxicity have been needed. One patient developed reversible hypophosphatemia and a second developed a benign skin lesion. 3 of 5 evaluable patients developed radioiodine uptake after 28 days of dabrafenib, and new radioiodine-avid lesions were demonstrated in all three after receiving a therapeutic dose of 131-I. All three patients demonstrated increases in thyroglobulin levels during treatment with dabrafenib. Conclusions: This initial data suggests that a subset of patients with radioiodine-resistant BRAF-mutant PTC demonstrate new iodine uptake following treatment with dabrafenib. Reuptake may correlate with increases in thyroglobulin, suggestive of re-differentiation. It is not yet known whether increased uptake of radioactive iodine will translate into a radiographic response. Two patients failed to convert to radioiodine-sensitive disease; it is possible that BRAF inhibition was incomplete in these patients and/or determinants other than BRAF mutation status contribute to radioiodine sensitivity. Clinical trial information: NCT01534897.

Thyroid cancer is the most common endocrine malignancy and it accounts for the largest fraction of endocrine cancer-related deaths. The accurate classification of well-differentiated thyroid tumors remains a significant challenge that impacts prognosis and therapeutic choices for patients. Papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) are the most common and the best characterized types of thyroid cancer, being diagnosed in approximately 80% and 15% of patients, respectively. Follicular variant of papillary thyroid carcinoma (FVPTC) is, after classical PTC, the most commonly diagnosed subtype of PTC, accounting for 9% to 22% of PTC cases. FVPTC exhibits some nuclear features of PTC but has a follicular growth pattern. Diagnosis of FVPTC presents a great challenge to pathologists, as these tumors can have distinct prognoses with variable clinical implications. While the incidence/diagnosis of FVPTC is growing in the U.S., the genetic characteristics of the disease remain poorly understood, and there is a need for complementary molecular assays that can help discriminate between the more benign forms of FVPTC and those that will exhibit an aggressive clinical course. In this study, we performed molecular analysis of 101 FVPTC archival surgical specimens using the SNaPshot multiplexed targeted sequencing platform, to interrogate 90 genetic loci frequently mutated in 21 cancer genes, including the BRAF, HRAS, KRAS, NRAS, PIK3CA and CTNNB1 (β-catenin) oncogenes commonly altered in thyroid cancer. Quantitative reverse transcriptase PCR (qRT-PCR) of cDNA obtained from the same tumors was used to test for the presence of RET/PTC1 and RET/PTC3 gene rearrangements found in PTC, and for PAX8-PPARγ gene fusions characteristic of FTC. We detected mutually exclusive genetic aberrations in 68 (67%) FVTPC. These included 25 (25%) BRAF V600E mutations, 26 (26%) mutations in NRAS (22 Q61R and 4 Q61K), 13 (13%) mutations in HRAS (9 Q61R, 3 Q61K, and 1 G13R) and 3 (3%) KRAS G12V mutations. We identified one (1%) single case of PAX8-PPARγ gene rearrangement and no RET/PTC gene fusions. Patient medical records and tumor histopathological characteristics will be retrospectively reviewed to determine whether the FVPTC genotype is significantly associated with clinicopathological features of diagnostic and prognostic importance that could help inform therapeutic decisions for this commonly diagnosed endocrine tumor. Citation Format: Dora Dias-Santagata, David G. McFadden, Lindsay A. Bernardo, Kerry D. Lynch, A. John Iafrate, Peter M. Sadow, Gilbert H. Daniels. Somatic mutation profiling of the follicular variant of papillary thyroid cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1191. doi:10.1158/1538-7445.AM2013-1191

Small cell lung carcinoma (SCLC) is a highly lethal tumor for which few targetable genetic alterations have been identified. SCLC is tightly associated with an extended history of tobacco use, and recent DNA sequencing studies have revealed highly mutated SCLC cancer genomes. Interestingly, two studies analyzing the human SCLC genome report distinct sets of putative driver mutations, highlighting the challenge of identifying functional events in highly mutated cancers. In the absence of environmental mutagens, a previously established genetically engineered mouse model of SCLC initiated by combined loss of Trp53 and Rb1 recapitulates the salient clinical features of human SCLC including tumor histologic progression and frequent distant metastases. This model has been shown to acquire DNA copy number alterations shared with human SCLC, suggesting that cross-species cancer genomics may identify a subset of driver events in human cancers. In order to characterize the spectrum of acquired events in murine SCLC and identify evolutionarily conserved drivers of SCLC progression, we define the somatic genome and transcriptome of a large panel of murine SCLC at single nucleotide resolution using exome, genome and RNA sequencing. We uncover complex subclonality in primary murine SCLC tumors and detect evidence for clonal selection during metastatic spread. As expected in the absence of environmental mutagens, we demonstrate a low somatic mutation frequency in murine SCLC. Using integrative genomic analyses we uncover alterations in chromatin remodeling enzymes and identify Pten as a critical tumor suppressor in this model. Engineered Pten deletion in murine SCLC dramatically accelerates tumorigenesis and fundamentally alters the genomic evolution of these tumors. This work represents the first large-scale comprehensive genomic characterization of a genetically engineered mouse cancer model at single nucleotide resolution, has implications for the nature of tumor evolution in mouse cancer models and identifies a potential therapeutic target in a subset of human SCLC. Citation Format: David G. McFadden, Thales Papagiannakopoulos, Kristian Cibulskis, Chip Stewart, Scott Carter, Amaro Taylor-Weiner, Arjun Bhutkar, Carrie Sougnez, Alison Dooley, Erica Shefler, Eric Lander, Stacey Gabriel, Gad Getz, Tyler Jacks. Comprehensive genomic analysis of murine small cell lung carcinoma uncovers recurrent Pten alterations that drive tumor progression and alter tumor genome evolution. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1084. doi:10.1158/1538-7445.AM2013-1084

Progression of lung adenocarcinoma to invasive, metastatic disease is responsible for the majority of deaths from lung cancer and remains incompletely understood. We have developed a genetically-engineered mouse model of lung adenocarcinoma initiated by mutant K-ras and loss of p53 that develops invasive, metastatic disease. In order to identify the genes and pathways that cooperate with oncogenic K-ras and loss of p53 to contribute to tumor development, progression, and metastasis, we have utilized in situ exon capture and massively parallel DNA sequencing to identify somatically-acquired point mutations in advanced stage murine lung adenocarcinoma cell lines. We have initially chosen a target set of 1283 cancer-relevant genes to develop our capture, sequencing, and bioinformatics platforms. Using tumor cell lines derived from K-ras-dependent tumors, we have successfully developed and refined these methods and have identified somatic mutations in our test samples. These mutations have been validated by conventional methods in the tumor cell line and founder tumor DNA. We have developed a production phase target gene set consisting of 1909 cancer-relevant genes and have initiated capture and sequencing of additional murine lung adenocarcinoma cell lines and tumors. We believe these datasets will expand the understanding of the biology of tumor progression and serve as an important biological filter for human cancer gene re-sequencing efforts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-151.

Summary Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Here, we describe the genomic landscape of 496 PTCs. We observed a low frequency of somatic alterations (relative to other carcinomas) and extended the set of known PTC driver alterations to include EIF1AX , PPM1D , and CHEK2 and diverse gene fusions. These discoveries reduced the fraction of PTC cases with unknown oncogenic driver from 25% to 3.5%. Combined analyses of genomic variants, gene expression, and methylation demonstrated that different driver groups lead to different pathologies with distinct signaling and differentiation characteristics. Similarly, we identified distinct molecular subgroups of BRAF -mutant tumors, and multidimensional analyses highlighted a potential involvement of oncomiRs in less-differentiated subgroups. Our results propose a reclassification of thyroid cancers into molecular subtypes that better reflect their underlying signaling and differentiation properties, which has the potential to improve their pathological classification and better inform the management of the disease.